Mitochondria have a variety of essential responsibilities in cellular metabolism, e.g. the production of ATP through oxidative phosphorylation in aerobic cells as well as the initiation of the signal cascade leading to apoptosis. Alteration of the mitochondrial proteome and altered mitochondrial function have been implicated in a variety of degenerative diseases, heart disease, aging, and cancer. Because the mitochondria are a major source of endogenously generated reactive oxygen species (ROS), it is not surprising that they have been proposed to play a major role in aging. The free radical theory of aging predicts that oxidative damage to the mitochondria can lead to an amplifying effect whereby damaged mitochondria release more ROS, further increasing oxidative damage. Considering the progressive age-dependent functional changes of most cell types and tissues, an accurate profile of age-dependent mitochondria proteome is mandatory and will shed light on the molecular mechanisms and pathways of age-associated disease processes. To greatly increase the mitochondria proteome coverage, particularly toward the identification of post-translational modifications (PTMs), this project aims to develop and demonstrate a capillary gel electrophoresis (CGE)-based multidimensional protein separation platform, capable of providing significant analyte concentration and extremely high resolving power for handling complex protein mixtures prior to mass spectrometry detection. In comparison with conventional SDS-PAGE-based proteome approaches, the proposed CGE/nano-reversed-phase liquid chromatography proteome technology will be highly automated and offer robust and high throughput fractionation of whole proteins for top-down proteomics while avoiding analyte dilution and loss in an integrated platform. By coupling with a nanoscale trypsin membrane reactor, the ultrafast proteolytic digestion of proteins resolved and eluted from the CGE-based separations enables the combined top-down/bottom-up characterization of PTMs in mouse liver mitochondria as the model system in this study.